Rapid molecular detection of clinically relevant yeasts by PCR-based denaturing high performance liquid chromatography

Abstract number: 1134_04_57

Goldenberg O., Graf B., Herrmann S., Adam T., Marjoram G., Fischer A., Hong G., Bereswill S., Göbel U.

Objectives:  

The detection and identification of clinically relevant yeast species by cultivation and biochemical testing is costly and time consuming. Thus, we established a rapid culture-independent molecular approach allowing both sensitive detection, as well as identification of individual yeast species.

Methods:  

Yeast DNA was detected in total DNA samples isolated from stool and from blood cultures, respectively, by amplification of the intergenic regions between ribosomal genes. Amplicons were separated by denaturing high-performance liquid chromatography (DHPLC) with the WAVE system (Transgenomics Inc.). Cultivation and biochemical testing of yeasts was performed in our diagnostics laboratory according to standard procedures.

Results:  

The analysis of yeast reference strains showed that a set of 13 different species could be identified by their DHPLC peak profiles and the corresponding retention times. The applicability of the method was evaluated by detection and identification of yeasts in clinical samples taken from complex matrices such as stool and blood. The peak-profiles and retention times obtained by DHPLC corresponded with the dominant yeast species in the samples, as confirmed by sequence analysis, cultivation and biochemical testing. If the retention times of amplicons from the samples were compared to standard strains in each run, the technique was reproducible, and valid data were obtained from blood cultures as well as from stool samples.

Conclusions:  

Detection and identification of yeast species in clinical specimens by PCR-based DHPLC complements the conventinal techniques available for yeast diagnostics.